Integrated 16S rRNA gene sequencing and LC/MS-based metabolomics ascertained synergistic influences of the combination of acupuncture and NaoMaiTong on ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Acupuncture is an effective therapy for ischemic stroke, which has been widely used in China and gradually accepted in more countries and regions recently. In addition, Chinese medicine also plays an important role in stroke treatment, among which NaoMaiTong (NMT) is an example of an effective herbal formula for the treatment of stroke. A therapeutic strategy that combines acupuncture and medicine was widely used in stroke patients. However, the synergistic influences and mechanisms of combined acupuncture and medicine on ischemic stroke have not yet been entirely elucidated. AIM OF THIS STUDY: The purpose of this study is to explore whether acupuncture and medicine combination treatments can produce synergism by using NMT, a clinically effective Chinese medicinal formula for the treatment of ischemic stroke for decades and has been demonstrated to be effective against ischemic brain injury, as a probe. Meanwhile, the potential mechanisms were investigated via cecal microbiome and plasma metabolomics to provide more strategies and basis for acupuncture-medicine combination for stroke. MATERIALS AND METHODS: Adopted middle-cerebral artery occlusion/reperfusion (MCAO/R) rat models, the effect for the stroke of the combination treatment consisting of acupuncture and NMT was evaluated by detecting neurological issues, cerebral infarct dimensions, levels of inflammatory factors (IL-6, IL-1ß, TNF-α) and oxidative stress factors (SOD, MDA) and brain-derived neurotrophic factor (BDNF). Subsequently,16S rRNA gene sequencing and LC/MS-based metabolomic analysis were utilized to explore the characteristics of cecal-contents microecology and plasma metabolic profile, respectively. Finally, the correlation between intestinal microecological characteristics and plasma metabolic characteristics was analyzed to explore the potential mechanism of the acupuncture-NMT combination. RESULTS: The efficacy of acupuncture-NMT therapy was more effective than a single treatment on ischemic stroke, with more effectively reduced infarct sizes, improved neurobehavioral deficits, and alleviated oxidative stress and inflammatory responses. Besides, the combination therapy not only adjusted gut microbiota disturbances by enriching species diversity, reducing the abundance of pathogenic bacteria (such as Escherichia-Shaigella), as well as increasing the abundance of beneficial bacteria (such as Turicibacter, Bifidobacterium), but also improved metabolic disorders by reversing metabolite plasma levels to normality. The results of the correlation analysis demonstrated a significant association between intestinal microbiota and plasma metabolic profile, especially the strong correlation of Turicibacter and isoflavones phyto-estrogens metabolites. CONCLUSION: The combination of acupuncture and NMT could produce synergism, suggesting acupuncture-medicine combination therapy might be more conducive to the recovery of ischemic stroke. And the potential mechanism was probably related to the mediation of intestinal microecology and plasma metabolism. Turicibacter and isoflavones phyto-estrogens metabolites might be the targets for acupuncture-NMT combination for stroke. Our current findings could provide a potential therapeutic strategy against ischemic stroke.

[Study on pharmacodynamic material basis of Naomaitong to protect neuronal cells based on PK-PD model].

The PK-PD correlation models by using pharmacodynamics and pharmacokinetics were applied to study the material basis of Naomaitong,a clinical empirical prescription for the treatment of cerebral apoplexy,in inhibiting the death of PC12 nerve cells induced by Na_2S_2O_4 and Glu. In this experiment,PC12 cell death models induced by Na_2S_2O_4 and Glu were established respectively.With LDH lateral leakage and NO content as pharmacodynamic indexes,PK-PD model was established by SVM algorithm to evaluate the effective components of Naomaitong in inhibiting neural cell death. The results showed that the positive correlation of emodin methyl ether-8-O-ß-D-glucopyranoside,aloe emodin,chrysophanol,rhein,emodin,ginsenoside Rg1,ginsenoside Rc,3'-methoxypuerarin and ligustilide was significant,obviously improving the LDH release and NO content. The results indicated that the contribution of Radix Puerariae Lobatae Radix and Rhei Radix et Rhizoma in Naomaitong could protect the nerve cell death induced by Na_2S_2O_4 and Glu respectively. PK-PD model was used to screen the neuroprotective components in Naomaitong,revealing the possible pharmacodynamic material basis of Naomaitong in the treatment of cerebral ischemia injury.

Study on pharmacodynamic material basis of Naomaitong to protect neuronal cells based on PK-PD model / 中国中药杂志

The PK-PD correlation models by using pharmacodynamics and pharmacokinetics were applied to study the material basis of Naomaitong,a clinical empirical prescription for the treatment of cerebral apoplexy,in inhibiting the death of PC12 nerve cells induced by Na_2S_2O_4 and Glu. In this experiment,PC12 cell death models induced by Na_2S_2O_4 and Glu were established respectively.With LDH lateral leakage and NO content as pharmacodynamic indexes,PK-PD model was established by SVM algorithm to evaluate the effective components of Naomaitong in inhibiting neural cell death. The results showed that the positive correlation of emodin methyl ether-8-O-β-D-glucopyranoside,aloe emodin,chrysophanol,rhein,emodin,ginsenoside Rg1,ginsenoside Rc,3'-methoxypuerarin and ligustilide was significant,obviously improving the LDH release and NO content. The results indicated that the contribution of Radix Puerariae Lobatae Radix and Rhei Radix et Rhizoma in Naomaitong could protect the nerve cell death induced by Na_2S_2O_4 and Glu respectively. PK-PD model was used to screen the neuroprotective components in Naomaitong,revealing the possible pharmacodynamic material basis of Naomaitong in the treatment of cerebral ischemia injury.

LC-high-resolution-MS/MS analysis of chemical compounds in rat plasma after oral administration of Nao-Mai-Tong and its individual herbs.

Nao-Mai-Tong (NMT), a Chinese herbal formula, is used for treating ischemia cerebral apoplexy. To discover the components of NMT with potential in vivo bioactivity and investigate the differences between NMT and its single herb components, this study rigorously compared plasma samples collected from rats dosed with NMT and single-herb extracts at different times through LC-high resolution-MS/MS and data processing (Metworks). The plasma of the NMT group contained a total of 66 identified compounds (25 prototypes and 41 metabolites), including anthraquinones, triterpenoid saponins, isoflavones and phthalides. Additionally, glucuronidation, sulfation and cysteine conjugation were the major reactions through which the compounds in NMT were metabolized. The comparison of the groups revealed two metabolites that were only detected in the plasma from the NMT-dosed group, whereas seven prototype ingredients (chrysophanol-8-O-glucoside, ginsenoside Rf, Rg2, Rh1, F1, F2 and chikusetsusaponin IVa) and 12 metabolites (two novel triterpenoid saponins) were only discovered in the plasma samples from the single-herb-dosed groups. Moreover, the trends in the chemical compounds detected presented marked differences between NMT-dosed rat plasma and plasma samples from the single-herb-dosed groups. The above data indicate that prescription compatibility affects the assimilation and elimination of ingredients and provides useful information for further pharmacokinetic studies.

Neuroprotective effect of Naomaitong extract following focal cerebral ischemia induced by middle cerebral artery occlusion in rats.

OBJECTIVE: To examine the neuroprotective effect of extract from Naomaitong following focal cerebral ischemia reperfusion induced by occlusion of middle cerebral artery (MCA), and to determine the biochemical alterations in urine using proton nuclear magnetic resonance spectroscopy and principal component analysis. METHODS: Wistar rats were randomly assigned to three groups: sham-operated group, MCA focal cerebral ischemia reperfusion model group, and active extract of Naomaitong treatment group. The model was established by an improved MCA occlusion (MCAO) method. Sham-operated rats received the same surgical procedure, but without occlusion. The Naomaitong treatment group were treated with active extract from Naomaitong at a dose of 3.0 gkg-1d-1. Brain tissues and urine samples were collected from all groups for histopathological assessment and proton nuclear magnetic resonance spectroscopy-based metabonomics, respectively. RESULTS: Hematoxylin-eosin and triphenyl tetrazolium chloride staining of brain tissues showed a significant decrease in cerebral infarction area in the Naomaitong group. In model rats, metabonomic analyses showed increased urinary levels of glutamate, taurine, trimetlylamine oxide, betaine, and glycine, and reduced levels of creatinine and creatine. Naomaitong regulated the metabolic changes by acting on multiple metabolic pathways, including glycine metabolism, glutaminolysis, transmethylation metabolism and creatinine metabolism. CONCLUSION: These data demonstrate that extract from Naomaitong is neuroprotective against focal cerebral ischemia induced by MCAO, and can alleviate biochemical changes in urinary metabolism. Metabonomics may be a useful approach for assessing the biochemical mechanisms underlying the neuroprotective actions of extract from Naomaitong.

Optimization of component compatibility of Naomaitong for anti-cerebral ischemia treatment based on artificial neural network / 中国药学杂志

OBJECTIVE: To optimize the component compatibility of Naomaitong for anti-cerebral ischemia treatment based on artificial neural network. METHODS: The five effective parts in Naomaitong were divided into ten groups by uniform design. Except the rats in the sham groups, all rats were subject to right middle cerebral artery occlusion (MCAO) with the suture-occluded method by Longa. The effect of Naomaitong was evaluated based on the bulk density of necrotic zone and infarction area percentage. The artificial neural network model was established for pharmacodynamic prediction. RESULTS: The model established in this study could predict the actions of different drug combinations. The best effect was obtained by the following formula: total anthraquinones of rhubarb (TAR) 180 mg·kg-1, total saponins of Ginseng (TSG) 70 mg·kg-1, total flavonoids of Pueraria (TFP) 450 mg·kg-1, total phenolic acid of Ligusticum wallichii (TPLW) 27 mg·kg-1, rhizome oil (RO) 110 μL·kg-1. CONCLUSION: Different compatibilities of Naomaitong are all effective for the treatment of ischemia in rats. Uniform design combined with ANN can be a more efficient method to realize dose optimization for Naomaitong prescription.

Study on effects of extract in Naomaitong Formula on cerebral ischemia-reperfusion model based on NMR metabolomics / 中草药

Objective: To clarify the effects of the extract in Naomaitong Formula (ENF) at different doses on cerebral ischemia-reperfusion model of rats and its mechanism in order to optimize the dose group. Methods The focal cerebral ischemia-reperfusion model of Wistar rats was established by an improved middle cerebral artery occlusion (MCAO) method. NMR and principal component analysis (PCA) were applied to analyzing the changes of rat plasma metabolites by ig administration of ENF at 1.5, 3.0, and 6.0 g/kg. And the treatment of ENF for cerebral ischemia-reperfusion model of rats was testified by HE staining with rat brain tissue biopsy. Results PCA and SPSS analyses indicated that the ENF at middle dose of 3.0 g/kg could inhibit the increase of glutamine, taurine, tyrosine, and lactic acid in the plasma of rats, promote the level of lipid, cholesterol, trihydroxy-butyric acid, N-acetyl aspartic acid, acetacetic acid, glutamic acid, and choline, and make them tend to normal. Conclusion Middle dose of ENF could efficiently regulate the metabolic balance after cerebral ischemia-reperfusion injury of rats, so as to play a role of resisting the cerebral ischemia-reperfusion injury of rats.

Content Determination of Ginsenosides in Naomaitong Granules by RP-HPLC Method / 中国中医药信息杂志

Objective To establish RP-HPLC method for determination of Ginsenosides in effective parts of Naomaitong Granules. Methods Using BDS C18 column (200 mm?4.6 mm, 5 ?m) of Dalian Elite, the mobile phase was a mixture of Acetonitrile-water gradient elution. The flow rate was 1.0 mL/min, the wavelength was 203 nm. Results The average recovery rate of Ginsenoside Rg1, Re and Rb1 was 100.71%, 100.14% and 100.94%, respectively, and RSD was 2.27%, 1.88% and 1.95%, respectively. Conclusion The method is simple, accurate and can be used to control the quality of Naomaitong Granules.

Adsorption of Active Components in Naomaitong Granules by Different Macroporous Adsorptive Resins / 中国药房

OBJECTIVE: To optimize the macroporous absorbing resin which were of best action in adsorption and desorption on the active components in Naomaitong granules. METHODS: UV spectrophotometry and HPLC was employed to determine the adsorbability and desorption capacity of different macroporous absorbing resins on total anthraquinones, total ginsenosides, total alkaloids and Puerarin. RESULTS: There were differences in adsorption and desorption capacity on active components in Naomaitong granules among different macroporous absorbing resins. Considering the general adsorbability and desorption capacity of different macroporous absorbing resins, AB-8 turned out to be of the best purification effect on Naomaitong granules. CONCLUSION: The results serve as a theoretical basis for the production of Naomaitong granules.

Content Determination of Total Saponins in Effective Composition of Naomaitong / 中国药房

OBJECTIVE:To establish the method for the content determination of total saponins in Naomaitong.METHODS:Colormetric method was applied using panoxadiol as control.The detection wavelength was set at 556 nm.RESULTS:The linear range of panoxadiol was 2.03～10.15 ?g?mL-1(r=0.999 6) with an average recovery of 100.69%(RSD=1.76%,n=6).CONCLUSION:The method is easy to operate with high precision,accuracy and good reproducibility for the quality control of Naomaitong.

Effects of Naomaitong on regulateing gene expression of gelatinase system after cerebal ischemia/reperfusion in aged rats / 中华中医药杂志

Objective: To study effects of Naomaitong on injury of cerebro-microvessel basement membrane in different periods and gene expression of gelatinase system after cerebral ischemia/reperfusion(I/R) in rats.Methods: Focal cerebral ischemia/reperfusion model was established by MCAO method of the intraluminal filament technique.Rats were divided into sham-operated group,model group,Naomaitong group and Nimodipine group at random,the later three groups with ischemia 3h and I/R 6h,12h,24h,3d,6d.Ransmission electron microscope and hybridization in situ method were used to observe the structure of micrangium and gene expression of gelatinase and its inhibitor.Results: Under electron microscope,the pathodamage in medico-group seemed lighter.According to the gene expression of gelatinase system,Naomaitong could step down the mRNA expression of MMP-2(I/R 6h-6d)and MMP-9(I/R 12h,I/R 3d).And increase the mRNA expression of TIMP-1(I/R 6h-3d).Conclusion: The protection of Naomaitong on cerebro-microvessel basement membrane after cerebral I/R in rats are related to the gene expression regulation of gelatinase system.

Protective effect and dosage-effect relationship of Naomaitong on inflammatory cascade response after focal cerebral ischemia reperfusion in aged rats / 中华中医药杂志

Objective: To study the protective e ects and dosage-e ect relationship of Naomaitong on focal cerebral ischemia reperfusion in aged rats.Methods : Focal cerebral ischemia model ( ischemia 3h and I/R12d)was duplicated with MCAO.The changes of the nervous dysfunction score ,the water content of cerebral constitution and the expression of TNF-?,VCAM-1,ICAM-1and its mRNA were observed. Results :The nervous dysfunction score ,the water content of cerebral constitution, the expression of TNF-?,VCAM-1,ICAM-1and its mRNA in aged model group were higher than those of the sham-operated group;but all of these were decreased in three Naomaitong groups and Nimodipine group compared with that of aged model group;the nervous dysfunction score and the expression of VCAM-1,ICAM-1 mRNA in Naomaitong group(0.9g?kg-1) were lighter than that of the Nimodipine group;the nervous dysfunction score,the water content of cerebral constitution, the expression of TNF-?,VCAM-1,ICAM-1and its mRNA in Naomaitong group(0.9g.g-1.d-1) were higher than that of Naomaitong group(0.45g?kg-1?d-1).Conclusion :Naomaitong could protect brain cell from damage after focal cerebral ischemia reperfusion injury by inhibiting the expression of TNF-?,VCAM-1,ICAM-1, with the middle dose of Naomaitong being more e ective.

Clinical Observation on Naomaitong Capsules for Treatment of 86 Cases of Cerebral Thrombosis / 中医杂志

Objective: To observe therapeutic effect of Naomaitong Capsules on cerebral thrombosis and to study the mechanism. Methods: 160 cases of thrombosis were randomly divided into a treatment group(n=86) and a control group(n=74), and they were treated by Naomaitong Capsules and LuoXinTong, respectively. After treatment for 3 months therapeutic effects were investigated. Results: The cured and markedly effective rate was 81.39% and the total effective rate was 95.35% in the treatment group with significant differences as compared with those in the control group (P

Variation of Ferulic Acid Content in Naomaitong by Different Extraction Process / 中药新药与临床药理

Objective To compare the variation of ferulic acid contents in Naomaitong by different extraction processes.Methods With ethanol or water as extracting solvent,the four herbal medicines of Rhizoma Chuanxiong,Radix Ginseng,Radix Puerariae,Radix et Rhizoma Rhei,which consisted of Naomaitong prescription,were decocted together or decocted separately firstly then mixed together were evaluated.With ferulic acid extraction rate as the index,the above extraction processes were evaluated.Results The extraction rate of ferulic acid extracted by alcohol was higher than that by water,but the ferulic acid content showed no obvious difference by decocting together or decocted separately firstly then mixed together.Conclusion It is suggested that proper extraction solvents and extraction methods should be adopted according to the different physicochemical characteristics of chemical contents in herbal medicine during extraction.

Content Determination of Gastrodine in Naomaito1g Oral Liquid with 2nd Derivative Spectrophotometry / 中成药

The 2nd derivative spectrophotometry was developed fo r the content determination of gastrodine in Naomaitong Oral Liquid. This methodis simple and rapid. Its' teproducibility is well.

Anthraquinones variation in extractive processes of Naomaitong extract / 中成药

AIM: To compare the contents variation of anthraquinones by 2 kinds of extractive process of Naomaitong extract (Radix et Rhizoma rhei, Rhizoma chuanxiong, Radix et Rhizoma qinseng, Radix puerariae lobatae, etc). METHODS: In application to RP-HPLC, Hypersil ODS2 C_ 18 column, the mobile phase was a mixture of methanol- 0.1% phosphate water (75 ∶25), the wavelength of detecting five aglycons in R. officinale was at 254 nm. RESULTS: The extraction rate of anthraquinones contents extracted by alcoholic extraction was higher than water extraction, the chrysophanol content in separated decoction was higher than that in the mixed decoction. CONCLUSION: There are dynamic variations in the extraction of Chinese medicine mixture. It is necessary to handle samples appropriately according to physichemical attributes of chemical contents.

Separation and purification of Naomaitong Granules by AB-8 macroporous absorption resin / 中成药

AIM: To study the purification of Naomaitong Granules(Radix et Rhizoma rhei,Rhizoma chuanxiong,Radix Puerariae lobatae.etc) by macroporous absorption resin. METHODS: Naomaitong Granule was purificated by macroporous absorption resin AB-8.UV spectrothotometry was used to determine the contents of total anthraquinones,total ginsenosides,total alkaloids,and the content of puerarin was determined by HPLC.The technic of purification was optimized according to the content above. RESULTS: The optimized technological conditions consisted of eoncentranon of original sample 120 mg/mL,the diameter and height was in proportion of 1∶10,the ratio of maximum adsorption to resin volume was 1∶6,water elution was 2B multiple of resin volume,8B multiple of resin volume 50% alcohol was the elution. CONCLUSION: AB-8 macroporous absorption resin can be used to purify Naomaitong Granules.

Correlation between fingerprint peaks and Compound Naomaitong 's effective fraction and its relevant herbs / 中成药

AIM:To establish the HPLC fingerprint of Compound Naomaitong effective parts,and to study the correlation analysis between fingerprint peaks and the effective fraction and its relevant herbs. METHODS:The chromatographic fingerprints of the effective fraction and the relevant fractions of its herbs were configured by HPLC/PDAD analysis. The relative deviation of retention time was utilized as indices to evaluate the correlation, the wavelength was set at 203 nm. RESULTS:The fingerprint of Compound Naomaitong effective parts was established and 36 copossessing fingerprint peaks were indicated. The assignment results of 14 peaks effective parts of fraction were indicated. CONCLUSION:The quality of Compound Naomaitong effective parts can be controlled by the HPLC fingerprint.